ESI-MS: Discussion of how this particular analytical technique is used in multiple chemistry fields.
Electrospray ionization mass spectrometry, also known as ESI-MS, is an analytic technique used to determine the molar weight of unknown compounds. Electrospray ionization is a soft technique whose main focus is to produce ions, which are then analyzed via mass spectrometry. [1] The phenomenon of electrospray was known to exist for several hundreds of years, but the theory behind it was not understood. It was not until the early 20th century that scientists began to fully understand how electrospray worked. Malcolm Dole, an American scientist, performed the pioneering experiment which used electrospray to ionize the chemical compounds he was working with.[2] ESI-MS technique was introduced in the 1980’s by Yamashita and John B. Fenn. In 2002, Fenn was awarded by the Royal Swedish Academy of Science the Nobel Prize in Chemistry for his introductory work on ESI-MS.[3]ESI-MS was recognized very soon after its discovery as a promising technique. [4] It became very popular in many science fields because it introduced a way to ionize ions without fragmenting them.
In order to start understanding electrospray ionization, it is necessary to become familiar with the components that make the ESI-MS instrument. Similar to electron and chemical ionization, electrospray ionization mass spectrometry has three major components; they include: ion sorter, mass analyzer and detector. In an ESI-MS instrument, the ion sorter, mass analyzer and detector are enclosed in distinct chambers where pressure is different; this enhances their functionality (see Fig.1). [5]
In the ion sorter chamber, the sample (dissolved in a polar volatile solvent), is injected by a mechanical syringe into a capillary. A high voltage of (2-6KV) is applied to the capillary, which causes the sample to turn into highly charge electrospray droplets. These droplets then move toward the mass spectrometer, directed by the flow of nitrogen gas. As the droplets are progressing toward the mass spectrometer, the aerosol droplets are reduced in size due to the solvent evaporating (see Fig.2). [6] Once the electrospray droplets make their way to the mass analyzer, charged plates direct the droplets to enter a heated capillary, which will help the aerosol reach the critical point, where they will undergo a Columbic explosion. The Columbic explosion will release the sample into the vapor phase. The ionization process that undergoes into the electrospray ionization mass spectrometry technique is what produces an intact molecular ion (with no fragmentation). [5]
Figure 2: Diagram showing the solution turning into electrospray droplets. Taken from [5]
The second step following the ion sorter is to analyze the ions produced. The mass spectrometry analyzes the sample by separating the different ions based on size, and then it sends them through the detector. There are many types of mass spectrometers that are used for this specific step. Some of them include, “magnetic (B)/electric (E) sector mass analyzer, linear quadrupole ion trap (LIT), three-dimensional quadrupole ion trap (QIT), orbitrap, time-of-flight mass analyzer (TOF) and ion cyclotron resonance mass analyzer (ICR).” [5]
The ions produced by the mass analyzer are detected by mass spectrometer. The mass spectrometer detects ions based on the mass per charge ration. After ions have been detected by the detector, then those signals are then converted, by a computer system, into peaks which are shown in a graph of m/z versus intensities. Figure 3 shows an example of how an ESI-MS spectrum looks like. [6] Some uncertainties exist about the height of the peaks seen in the graph. Precise information does not exist with respect to whether the peaks shown number of charge, or the number of ions present. [5]
Figure 3: ESI-MS spectrum of glycoprotein molecule. First graph shows m/z versus relative intensity, while second graph gives mass (in Dalton) versus relative intensity. Taken from [7]
As mentioned previously, electrospray ionization mass spectrometry is a widely used technique ever since it was presented. Scientists all over the world used ESI-MS for characterization of large size molecule, such as proteins. But, besides studying proteins, other fields such as organic chemistry and inorganic chemistry are also using this technique quite a bit. On one hand, organic chemists have found ESI-MS useful in determining multiple functional groups, assessing the purity of compounds, and analyzing absorption, distribution, and the metabolic pathway of drug molecules.[7] On the other hand, inorganic chemists use electrospray ionization mass spectrometry to determine the metal bound to ligands and more specifically they are able to determine the configuration of the metal.[8]
In the field of organic chemistry, ESI-MS technique is widely used for synthetic drug characterization. In an article published in Analytical Chemistry Journal researchers have used electrospray ionization mass spectrometry to characterize drugs containing two different functional groups. Those two functional groups include: amine containing side chains and nitrogen-containing saturated ring structures. In the particular case of drugs with amine-containing side chains, it was discovered that the amine group would be the first group to be cleaved followed by an alkene group (see Fig. 4). This particular pattern was suggested by one of the scientists in the article, and the data was supported by ESI-MS. The same pattern was compared against the electron-impact mass spectrometry (EI-MS), and the data did not correspond at all, showing no molecular ion present. In the case of drugs with nitrogen-containing saturated ring structures, it was determined by ESI-MS that the first functional groups cleaved from a molecule depends greatly on the higher negative heat of formation (see Figure 5). This data can be applied to heterocyclic compounds with amine functional group. [3]
Electrospray ionization mass spectrometer has been also used to characterize the fragmentation pathway of alkaloid nicotine molecules and its metabolites. Nicotine is a naturally occurring molecule found in the family of Solanaceae plants. It is widely used in tobacco products due to its addictive nature and it is found to make up 0.3 to 5 percent of its dry weight. Nicotine has been also found in very small quantity in vegetables such as potatoes, eggplants and green peppers. [9] Nicotine acts as a poison and it has been shown to increase the flow of dopamine, a neurotransmitter, in the brain, creating pleasure feeling. [10] Studies of nicotine by electrospray ionization MS has shown the pseudo molecular ion as well as different metabolites seen as fragments. Figure 6 shows some fragmentation pattern of nicotine. [3]
Electrospray ionization mass spectrometry has also been used in characterizing and identifying polyoxometalates. Polyoxometalates (POM) are molybdenum/ tungsten/ vanadium/niobium metal oxide cluster structures known for having interesting physical and chemical properties. The chemistry of small POM’s is known because small POM structures have been identified. The problem lies into not knowing the structure of large POM molecules; large POM clusters range from 10- 16 KDa. These POM clusters are known as Wells-Dawson clusters and they are famous due to their ability to accept many electrons without any structural modification. A lot of literature does not exist on Wells-Dawson clusters because of the difficulties that exist with the characterization of such huge molecules. Well known analytical techniques, such as NMR spectroscopy, polarography, optical spectrophotometry, among many other techniques are not very useful in identifying large molecules. So knowing this, scientists have found one way to determine the structure of large POM cluster by developing an inorganic and organic hybrid structure. In this experiment, a hybrid POM molecule was used; the hybrid molecule was developed by combining clusters with organic compounds. The technique used to characterize and identify hybrid cluster was ESI-MS. Through this research, it was found that electrospray ionization mass spectroscopy was a valuable tool. When a sample was analyzed by ESI-MS, the data obtained revealed group of peaks pertaining to different anions. Many Wells-Dawson hybrid clusters were analyzed and all their ESI-MS spectra showed clear and simple peaks; this is thought to be a result of non-fragmentation of the original molecule. A good example of this is seen in Figure 7. In the spectra seen below, there are peaks with different anionic charge states, ranging from -3 to -6. [8]
Similar to characterizing POM compounds by electrospray ionization mass spectrometry, ESI-MS has also been used in identifying inorganic molecules. The field of inorganic chemistry is involved in studying inorganic compound such as organometallics, which have an important role in biological processes. [11] Organometallics are compounds that have organic molecules bonded to a metal. The importance of metals and their ligand forming properties, make scientists more eager to know their molecular weight as well as their structure. Analytical techniques such as X-ray crystallography and NMR spectroscopy are common ways used to characterize these complexes. Ever since electrospray ionization mass spectroscopy was introduced, it has become a major technique to determine the exact molecular weight of these complexes. ESI-MS is widely used in organometallics because of its ability to give the molecular ion. In an experiment done by an Argentinian research group, rhenium complexes were analyzed with multiple analytical techniques. As a result, ESI-MS was able to detect the molecular ion of some complexes; some peaks due to fragmentation were also seen. As seen in Table 1, for most complexes the molecular ion is clearly visible, and easily detected. From table 1, it is seen that electrospray ionization gave the most molecular ions from all the other analyzing methods. For the first three rhenium compounds, ESI gave fragmented molecular ion, and when compared to the results from probe electrospray ionization (PESI), similar results were seen. The difference is that in PESI, an additional H+ was shown. For the third compound, laser desorption ionization (LDI) and matrix-assisted laser desorption ionization (MALDI) were the only two techniques that gave [M]-. [12]
Compared to organometallics, proteins are among the most used as samples for electrospray ionization mass spectrometry. There are many reasons that biochemists use ESI-MS technique. Besides finding molecular weight and clues about structure of proteins, ESI-MS is mostly used as an inborn screening technology in clinical setting. [6] One of the great benefits that ESI-MS provides for proteins is that it analyzes proteins that have very large molecular weight; larger than the range of the mass spectrometer. Also, electrospray is a soft ionization technique which does not fragment the proteins as much as the other available techniques. Below, it is shown the ESI-MS spectra of myoglobin; myoglobin is a protein related to hemoglobin, which bind to iron and oxygen. Myoglobin, like many proteins is very big, around 17 KDa. [13]
Amino acids are also being analyzed by ESI-MS. Some researchers in Italy have developed high-performance liquid chromatography / electrospray ionization mass spectrometry (HPLC-ESI-MS/MS) to analyze all underivatized amino acids. Amino acids are the building blocks of proteins and their presence in the human body is vital. If an amino acid is missing or malfunctioning, could lead to protein malfunctioning or enzyme deficiencies. This ultimately leads to diseases that are difficult to cure. [14] Medicine nowadays, is trying to take a step toward preventative measures, because it is easier and better to prevent a disease rather than curing.[15] Scientists together with doctors are moving into finding way to prevent getting a disease. A research group in Italy is using HPLC-ESI-MS/MS to screen newborn babies by using drops of their blood to determine the amino acid concentration levels. HPLC-ESI-MS/MS is a new developed method which makes it very fast and simple to analyze underivated amino acids from few blood spots. Unlike HPLC-ESI-MS/MS technique, other amino acid separation techniques require pre or post column derivatization to enhance the detection. The results of the experiment showed, that only six out of forty underivatized amino acids showed a weak signal. The rest of the amino acids showed a strong signal. The purpose of this experiment and the data they received is important because it indicated the deficiency cause by the amount of amino acids present in the newborn, which helps doctors into predicting a particular disease. All the predictions that the group made with respect to some infants were correct, which leads into believing that this new method is providing useful data.[14]
Unlike the research group in Italy, other researchers are using plain electrospray ionization mass spectroscopy to detect inborn errors of metabolism (IEM). IEM disorders are found to be prevalent in segregated communities, and it discovered that 1 in every 5000 births is at risk for IEM. In the field of clinical chemistry, ESI-MS is used because of its ability to be specific and give accurate results. Many researchers are working into developing the best way to prepare the blood-spot samples, that would detect abnormal parameters of amino acids in newborn.[16]
ESI-MS has also found its use in the food industry. Because of population increase in the world, the demand for oil, in particular palm oil is increasing as well. In 2006, it was found that 33% of all the oil used came from palm oil.[17]Malaysia is the leading producer of palm oil, and the high demand for it, is creating a concern about the environmental impact in the country. Therefore, scientists are now looking at ways to turn palm oil waste into consumable products. Scientists are putting a lot of time into working with PKC because even though it is a byproduct, it has been discovered that PKC contains 16-19% residual protein. Data and experiments have shown that there is a huge possibility of PKC being a potential source of peptides, with many biological functions, such as carotenes, tocopherols, phospholipids, coenzymes and phenolic acid. Research with mice has also shown that PKC peptides have an anti-fatigue effect on mice. Additional benefits of palm kernel cake have been linked to antimicrobial activity toward Bacillus species. These important factors are playing a huge role into getting the food and pharmaceutical companies involved into determining the composition of newly protein products made from PKC. Many biochemical analyses are being used to get more information about PKC, but ESI was chosen to characterize the antibacterial PKC compound. ESI is very sensitive method to analyzing big molecules such as proteins. From the electrospray ionization mass spectrometry results, it was determined that the major components of the sample analyzed had a signal around m/z 199.2 and through data comparison, it was determine to lauric acid, which has a signal m/z of 200.3 Da. Finding lauric acid in the sample was not surprising for the researchers because of the fact that lauric acid is the predominant components in palm oil. The additional information provided by ESI-MS was that the protein was not pure peptides but instead it was a complex peptide with a large amount of solvent derivative. The data received from electrospray was not conclusive on the peptide; it gave information about the purity of the complex.[18] The next step that any scientist at this time would take, would be to use other techniques to verify the structure of the peptide.
Many times in scientific fields, only one instrument will not solve the whole mystery about one compound. In many cases, working with multiple instruments will facilitate the work into figuring out the complete structure and fragmentation pathways of compounds. Laser electrospray mass spectroscopy is one the newly developed technique using ESI-MS, which is useful in many field. In one case, LEMS was used to for the determination of inorganic explosive devices. [19] An additional new technology involving ESI-MS, is the electrospray ionization time-of-flight mass spectrometry, which is widely used in metal complexes.[20]
A research group in Pakistan worked on identifying and characterizing the structures of multiple steroidal alkaloids in Sarcococca coriacea. Sarcococca coriacea is a plant from the genus Sarcococca, which are widely used in Africa and Asia as medicinal plant for treatment of pain and many other diseases. The Pakistani research group characterized 23 pregnane-type steroidal alkaloids by using electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS). This technique is a hybrid technique, where for the first step electrospray ionization quadrupole time-of-flight mass spectrometry was used to protonate the nucleus forming the molecular ion. In the second step, low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) was used to point the fragment, which shows the loss of dimethylamine. The data of the experiment did in fact show the observed mass of all 23 pregnane-type steroidal alkaloids, and when compared to known values, the percent errors were single digits. Using CID-MS/MS was extremely helpful into generating data that are useful for proposing a fragmentation pathway for the functional groups (see figure 11). [21]
Taken from [21]
Taken from [21]
Similar to every analytical technique in the world today, no single technique is able to provide answers about the characterization of a compound. ESI-MS has some advantages and some disadvantage. Electrospray ionization is a soft ionization technique which does not use a lot of energy, and as a result the sample being analyzes does not undergo through many fragmentations.[4] This is an advantage as more scientists are looking for instruments that will give them some conclusive information about the molecular weight of the large molecule. This leads to the second advantage is that a molecular ion is visible and it is so because ESI-MS is highly sensitive with good specificity.[5]Even though ESI-MS gives information about proteins such as molecular ions, there are cases where getting the molecular ion of some long proteins is not so easy. There have been reported cases where the molecular ion of some proteins were not visible, and that lowers the use of ESI-MS. Another disadvantage of ESI-MS is on the detection of peaks corresponding to dimeric species; conclusive evidence cannot be made by ESI-MS to show if dimeric species are present or not in a sample. [22] Also, in some sample, the peaks were not strong signals and that draws scientists a little back, as they are not sure about the nature of the peak. So, after stating the advantages and disadvantage of ESI-MS, it can be said that the technique is very useful in the characterization of compounds. To enhance this technique many scientists are combining this technique to other ones for better results.
In conclusion, electrospray ionization is the most commonly used analytical technique used by chemists and biochemist. In this paper, many examples of the use of ESI-MS in different fields have been reported to introduce the many different ways that this technique has been adopted. It is believed by many scientists that ESI-MS is still maturing, which leads us into hoping that someday, in the near future, ESI-MS can helps us identify and characterize with higher precision different types of compounds.[22] Electrospray ionization mass spectroscopy, similar to other analytical technique has both advantages and disadvantages to it, but looking at the information is has helped us gain so far, I strongly believe that its advantages overcome disadvantages. We are able to see its contributing when it is combined with other technique such as quadrupole time-of-flight, HPLC, and MS/MS. [6]
3) Smyth, W.F., McClean, S., Hack, C.J., Ramachandran, V.N., Doherty, B., Joyce, C., O’Donnell, F., Smyth, T.J., O’Kane, E. and Brooks, P.,“The characterisation of synthetic and natural-product pharmaceuticals by electrospray ionisation-mass spectrometry (ESI-MS) and liquid chromatography (LC)-ESI-MS.”TrAC Trends in Analytical Chemistry, 2006. 25(6), pp. 572-582. [http://dx.doi.org/10.1016/j.trac.2006.04.003]
4) Banerjee, S., Mazumdar, S., 2011. "Electrospray Ionization Mass Spectrometry: A Technique to Access the Information beyond the Molecular Weight of the Analyte,”International Journal of Analytical Chemistry2006.<http://www.hindawi.com/journals/ijac/2012/282574/>
6) Ho, C. S., Chan, M.H.M., Cheung, R.C.K., Law, L.K., Lit, L.C.W., Ng, K.F., Suen, M.W.M., Tai, H.L. "Electrospray Ionisation Mass Spectrometry: Principles and Clinical Applications." The Clinical Biochemistry Reviews 12th ser. 24.3 (2003): n. pag.NCBI. Web. 7 Dec. 2012. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1853331/>.
7) Martin, L., Perret, S., Kelly, J., Boulais, D., Cass, B., Bisson, L., Afkhamizarreh, F., Durocher, Y., "Stable High Volumetric Production of Glycosylated Human Recombinant IFNalpha2b in HEK293 Cells." 2008): BMC Biotechnology. Springer, 27 Aug. 2008. Web. 7 Dec. 2012. <http://www.biomedcentral.com/1472-6750/8/65>. [DOI:10.1186/1472-6750-8-65]
8)Pradeep, C.P., Li, F., Lydon, C., Miras, H.N., Long, D., Xu, L., Cronin, L. “Design and Synthesis of "Dumb-bell" and "Triangular" Inorganic-Organic Hybrid Nanopolyoxometalate Clusters and Their Characterisation through ESI-MS Analyses.” Chemistry-a European Journal, 2011. 17(27), pp. 7472-7479. [DOI: 10.1002/chem.201100257]
12) Petroselli, G., Mandal, M.K., Chen, L.C., Ruiz, G.T., Wolcan, E., Hiraoka, K., Nonami, H. and Erra-Balsells, R. “Mass spectrometry of rhenium complexes: a comparative study by using LDI-MS, MALDI-MS, PESI-MS and ESI-MS.” Journal of Mass Spectrometry, 2012.47(3), pp. 313-321. [DOI: 10.1002/jms.2965]
14) Zoppa, M., Gallo, L., Zacchello, F. and Giordano, G. “Method for the quantification of underivatized amino acids on dry blood spots from newborn screening by HPLC-ESI-MS/MS.” Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2006.831(1-2), pp. 267-273. [http://dx.doi.org/10.1016/j.jchromb.2005.12.015]
16) Rashed, Mohamed S. "Screening Blood Spots for Inborn Errors of Metabolism by Electrospray Tandem Mass Spectrometry with a Microplate Batch Process and a Computer Algorithm for Automated Flagging of Abnormal Profiles." Molecular Pathology 43.7 (1997): n. pag. Clinical Chemistry. Web. 7 Dec. 2012. <http://www.clinchem.org/content/43/7/1129.full>.
17) Chai, S.S., & Mohamed, A.R. "Utilization of oil palm as a source of renewable energy in Malaysia." Renewable and Sustainable Energy Reviews. 2006. 12, pp.2401-2421. [http://dx.doi.org/10.1016/j.rser.2007.06.006]
18) Tan, Y.N., Ayob, M.K., Yaacob, W.A.W. “Purification and characterisation of antibacterial peptide-containing compound derived from palm kernel cake.” Food Chemistry,2013.136(1), pp. 279-284. [DOI: 10.1016/j.foodchem.2012.08.012 ]
19) Flanigan, P.M., Brady, J.J., Judge, E.J. and Levis, R.J. “Determination of Inorganic Improvised Explosive Device Signatures Using Laser Electrospray Mass Spectrometry Detection with Offline Classification.” Analytical Chemistry, 2011.83(18), pp. 7115-7122. [DOI:10.1021/ac2014299]
20)Rellan-Alvarez, R., Abadia, J. and Alvarez-Fernandez, A. “Formation of metal-nicotianamine complexes as affected by pH, ligand exchange with citrate and metal exchange. A study by electrospray ionization time-of-flight mass spectrometry.” Rapid Communications in Mass Spectrometry, 2008.22(10), pp. 1553-1562. [DOI: 10.1002/rcm.3523]
21)Musharraf, S. G., Goher, M.,Ali, A., Adhikari, A. "Rapid Characterization and Identification of Steroidal Alkaloids in Sarcococca Coriacea using Liquid Chromatography Coupled with Electrospray Ionization Quadropole Time-of-Flight Mass Spectrometry."Steroids 77.1–2 (2012): 138-48. Print. [http://dx.doi.org/10.1016/j.steroids.2011.11.001]
22) Schug, K.A., Serrano, C., Frycak, P. “Controlled Band Dispersion For Quantitative Binding Determination and Analysis with Electrospray Ionization-Mass Spectrometry” Mass spectrometry reviews, 2011.30(1), pp. 176-176. [DOI: 10.1002/mas.20267]
Electrospray ionization mass spectrometry, also known as ESI-MS, is an analytic technique used to determine the molar weight of unknown compounds. Electrospray ionization is a soft technique whose main focus is to produce ions, which are then analyzed via mass spectrometry. [1] The phenomenon of electrospray was known to exist for several hundreds of years, but the theory behind it was not understood. It was not until the early 20th century that scientists began to fully understand how electrospray worked. Malcolm Dole, an American scientist, performed the pioneering experiment which used electrospray to ionize the chemical compounds he was working with.[2] ESI-MS technique was introduced in the 1980’s by Yamashita and John B. Fenn. In 2002, Fenn was awarded by the Royal Swedish Academy of Science the Nobel Prize in Chemistry for his introductory work on ESI-MS.[3]ESI-MS was recognized very soon after its discovery as a promising technique. [4] It became very popular in many science fields because it introduced a way to ionize ions without fragmenting them.
In order to start understanding electrospray ionization, it is necessary to become familiar with the components that make the ESI-MS instrument. Similar to electron and chemical ionization, electrospray ionization mass spectrometry has three major components; they include: ion sorter, mass analyzer and detector. In an ESI-MS instrument, the ion sorter, mass analyzer and detector are enclosed in distinct chambers where pressure is different; this enhances their functionality (see Fig.1). [5]
In the ion sorter chamber, the sample (dissolved in a polar volatile solvent), is injected by a mechanical syringe into a capillary. A high voltage of (2-6KV) is applied to the capillary, which causes the sample to turn into highly charge electrospray droplets. These droplets then move toward the mass spectrometer, directed by the flow of nitrogen gas. As the droplets are progressing toward the mass spectrometer, the aerosol droplets are reduced in size due to the solvent evaporating (see Fig.2). [6] Once the electrospray droplets make their way to the mass analyzer, charged plates direct the droplets to enter a heated capillary, which will help the aerosol reach the critical point, where they will undergo a Columbic explosion. The Columbic explosion will release the sample into the vapor phase. The ionization process that undergoes into the electrospray ionization mass spectrometry technique is what produces an intact molecular ion (with no fragmentation). [5]
Figure 2: Diagram showing the solution turning into electrospray droplets. Taken from [5]
The second step following the ion sorter is to analyze the ions produced. The mass spectrometry analyzes the sample by separating the different ions based on size, and then it sends them through the detector. There are many types of mass spectrometers that are used for this specific step. Some of them include, “magnetic (B)/electric (E) sector mass analyzer, linear quadrupole ion trap (LIT), three-dimensional quadrupole ion trap (QIT), orbitrap, time-of-flight mass analyzer (TOF) and ion cyclotron resonance mass analyzer (ICR).” [5]
The ions produced by the mass analyzer are detected by mass spectrometer. The mass spectrometer detects ions based on the mass per charge ration. After ions have been detected by the detector, then those signals are then converted, by a computer system, into peaks which are shown in a graph of m/z versus intensities. Figure 3 shows an example of how an ESI-MS spectrum looks like. [6] Some uncertainties exist about the height of the peaks seen in the graph. Precise information does not exist with respect to whether the peaks shown number of charge, or the number of ions present. [5]
Figure 3: ESI-MS spectrum of glycoprotein molecule. First graph shows m/z versus relative intensity, while second graph gives mass (in Dalton) versus relative intensity. Taken from [7]
As mentioned previously, electrospray ionization mass spectrometry is a widely used technique ever since it was presented. Scientists all over the world used ESI-MS for characterization of large size molecule, such as proteins. But, besides studying proteins, other fields such as organic chemistry and inorganic chemistry are also using this technique quite a bit. On one hand, organic chemists have found ESI-MS useful in determining multiple functional groups, assessing the purity of compounds, and analyzing absorption, distribution, and the metabolic pathway of drug molecules.[7] On the other hand, inorganic chemists use electrospray ionization mass spectrometry to determine the metal bound to ligands and more specifically they are able to determine the configuration of the metal.[8]
In the field of organic chemistry, ESI-MS technique is widely used for synthetic drug characterization. In an article published in Analytical Chemistry Journal researchers have used electrospray ionization mass spectrometry to characterize drugs containing two different functional groups. Those two functional groups include: amine containing side chains and nitrogen-containing saturated ring structures. In the particular case of drugs with amine-containing side chains, it was discovered that the amine group would be the first group to be cleaved followed by an alkene group (see Fig. 4). This particular pattern was suggested by one of the scientists in the article, and the data was supported by ESI-MS. The same pattern was compared against the electron-impact mass spectrometry (EI-MS), and the data did not correspond at all, showing no molecular ion present. In the case of drugs with nitrogen-containing saturated ring structures, it was determined by ESI-MS that the first functional groups cleaved from a molecule depends greatly on the higher negative heat of formation (see Figure 5). This data can be applied to heterocyclic compounds with amine functional group. [3]
Electrospray ionization mass spectrometer has been also used to characterize the fragmentation pathway of alkaloid nicotine molecules and its metabolites. Nicotine is a naturally occurring molecule found in the family of Solanaceae plants. It is widely used in tobacco products due to its addictive nature and it is found to make up 0.3 to 5 percent of its dry weight. Nicotine has been also found in very small quantity in vegetables such as potatoes, eggplants and green peppers. [9] Nicotine acts as a poison and it has been shown to increase the flow of dopamine, a neurotransmitter, in the brain, creating pleasure feeling. [10] Studies of nicotine by electrospray ionization MS has shown the pseudo molecular ion as well as different metabolites seen as fragments. Figure 6 shows some fragmentation pattern of nicotine. [3]
Electrospray ionization mass spectrometry has also been used in characterizing and identifying polyoxometalates. Polyoxometalates (POM) are molybdenum/ tungsten/ vanadium/niobium metal oxide cluster structures known for having interesting physical and chemical properties. The chemistry of small POM’s is known because small POM structures have been identified. The problem lies into not knowing the structure of large POM molecules; large POM clusters range from 10- 16 KDa. These POM clusters are known as Wells-Dawson clusters and they are famous due to their ability to accept many electrons without any structural modification. A lot of literature does not exist on Wells-Dawson clusters because of the difficulties that exist with the characterization of such huge molecules. Well known analytical techniques, such as NMR spectroscopy, polarography, optical spectrophotometry, among many other techniques are not very useful in identifying large molecules. So knowing this, scientists have found one way to determine the structure of large POM cluster by developing an inorganic and organic hybrid structure. In this experiment, a hybrid POM molecule was used; the hybrid molecule was developed by combining clusters with organic compounds. The technique used to characterize and identify hybrid cluster was ESI-MS. Through this research, it was found that electrospray ionization mass spectroscopy was a valuable tool. When a sample was analyzed by ESI-MS, the data obtained revealed group of peaks pertaining to different anions. Many Wells-Dawson hybrid clusters were analyzed and all their ESI-MS spectra showed clear and simple peaks; this is thought to be a result of non-fragmentation of the original molecule. A good example of this is seen in Figure 7. In the spectra seen below, there are peaks with different anionic charge states, ranging from -3 to -6. [8]
Similar to characterizing POM compounds by electrospray ionization mass spectrometry, ESI-MS has also been used in identifying inorganic molecules. The field of inorganic chemistry is involved in studying inorganic compound such as organometallics, which have an important role in biological processes. [11] Organometallics are compounds that have organic molecules bonded to a metal. The importance of metals and their ligand forming properties, make scientists more eager to know their molecular weight as well as their structure. Analytical techniques such as X-ray crystallography and NMR spectroscopy are common ways used to characterize these complexes. Ever since electrospray ionization mass spectroscopy was introduced, it has become a major technique to determine the exact molecular weight of these complexes. ESI-MS is widely used in organometallics because of its ability to give the molecular ion. In an experiment done by an Argentinian research group, rhenium complexes were analyzed with multiple analytical techniques. As a result, ESI-MS was able to detect the molecular ion of some complexes; some peaks due to fragmentation were also seen. As seen in Table 1, for most complexes the molecular ion is clearly visible, and easily detected. From table 1, it is seen that electrospray ionization gave the most molecular ions from all the other analyzing methods. For the first three rhenium compounds, ESI gave fragmented molecular ion, and when compared to the results from probe electrospray ionization (PESI), similar results were seen. The difference is that in PESI, an additional H+ was shown. For the third compound, laser desorption ionization (LDI) and matrix-assisted laser desorption ionization (MALDI) were the only two techniques that gave [M]-. [12]
Compared to organometallics, proteins are among the most used as samples for electrospray ionization mass spectrometry. There are many reasons that biochemists use ESI-MS technique. Besides finding molecular weight and clues about structure of proteins, ESI-MS is mostly used as an inborn screening technology in clinical setting. [6] One of the great benefits that ESI-MS provides for proteins is that it analyzes proteins that have very large molecular weight; larger than the range of the mass spectrometer. Also, electrospray is a soft ionization technique which does not fragment the proteins as much as the other available techniques. Below, it is shown the ESI-MS spectra of myoglobin; myoglobin is a protein related to hemoglobin, which bind to iron and oxygen. Myoglobin, like many proteins is very big, around 17 KDa. [13]
Amino acids are also being analyzed by ESI-MS. Some researchers in Italy have developed high-performance liquid chromatography / electrospray ionization mass spectrometry (HPLC-ESI-MS/MS) to analyze all underivatized amino acids. Amino acids are the building blocks of proteins and their presence in the human body is vital. If an amino acid is missing or malfunctioning, could lead to protein malfunctioning or enzyme deficiencies. This ultimately leads to diseases that are difficult to cure. [14] Medicine nowadays, is trying to take a step toward preventative measures, because it is easier and better to prevent a disease rather than curing.[15] Scientists together with doctors are moving into finding way to prevent getting a disease. A research group in Italy is using HPLC-ESI-MS/MS to screen newborn babies by using drops of their blood to determine the amino acid concentration levels. HPLC-ESI-MS/MS is a new developed method which makes it very fast and simple to analyze underivated amino acids from few blood spots. Unlike HPLC-ESI-MS/MS technique, other amino acid separation techniques require pre or post column derivatization to enhance the detection. The results of the experiment showed, that only six out of forty underivatized amino acids showed a weak signal. The rest of the amino acids showed a strong signal. The purpose of this experiment and the data they received is important because it indicated the deficiency cause by the amount of amino acids present in the newborn, which helps doctors into predicting a particular disease. All the predictions that the group made with respect to some infants were correct, which leads into believing that this new method is providing useful data.[14]
Unlike the research group in Italy, other researchers are using plain electrospray ionization mass spectroscopy to detect inborn errors of metabolism (IEM). IEM disorders are found to be prevalent in segregated communities, and it discovered that 1 in every 5000 births is at risk for IEM. In the field of clinical chemistry, ESI-MS is used because of its ability to be specific and give accurate results. Many researchers are working into developing the best way to prepare the blood-spot samples, that would detect abnormal parameters of amino acids in newborn.[16]
ESI-MS has also found its use in the food industry. Because of population increase in the world, the demand for oil, in particular palm oil is increasing as well. In 2006, it was found that 33% of all the oil used came from palm oil.[17] Malaysia is the leading producer of palm oil, and the high demand for it, is creating a concern about the environmental impact in the country. Therefore, scientists are now looking at ways to turn palm oil waste into consumable products. Scientists are putting a lot of time into working with PKC because even though it is a byproduct, it has been discovered that PKC contains 16-19% residual protein. Data and experiments have shown that there is a huge possibility of PKC being a potential source of peptides, with many biological functions, such as carotenes, tocopherols, phospholipids, coenzymes and phenolic acid. Research with mice has also shown that PKC peptides have an anti-fatigue effect on mice. Additional benefits of palm kernel cake have been linked to antimicrobial activity toward Bacillus species. These important factors are playing a huge role into getting the food and pharmaceutical companies involved into determining the composition of newly protein products made from PKC. Many biochemical analyses are being used to get more information about PKC, but ESI was chosen to characterize the antibacterial PKC compound. ESI is very sensitive method to analyzing big molecules such as proteins. From the electrospray ionization mass spectrometry results, it was determined that the major components of the sample analyzed had a signal around m/z 199.2 and through data comparison, it was determine to lauric acid, which has a signal m/z of 200.3 Da. Finding lauric acid in the sample was not surprising for the researchers because of the fact that lauric acid is the predominant components in palm oil. The additional information provided by ESI-MS was that the protein was not pure peptides but instead it was a complex peptide with a large amount of solvent derivative. The data received from electrospray was not conclusive on the peptide; it gave information about the purity of the complex.[18] The next step that any scientist at this time would take, would be to use other techniques to verify the structure of the peptide.
Many times in scientific fields, only one instrument will not solve the whole mystery about one compound. In many cases, working with multiple instruments will facilitate the work into figuring out the complete structure and fragmentation pathways of compounds. Laser electrospray mass spectroscopy is one the newly developed technique using ESI-MS, which is useful in many field. In one case, LEMS was used to for the determination of inorganic explosive devices. [19] An additional new technology involving ESI-MS, is the electrospray ionization time-of-flight mass spectrometry, which is widely used in metal complexes.[20]
A research group in Pakistan worked on identifying and characterizing the structures of multiple steroidal alkaloids in Sarcococca coriacea. Sarcococca coriacea is a plant from the genus Sarcococca, which are widely used in Africa and Asia as medicinal plant for treatment of pain and many other diseases. The Pakistani research group characterized 23 pregnane-type steroidal alkaloids by using electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS). This technique is a hybrid technique, where for the first step electrospray ionization quadrupole time-of-flight mass spectrometry was used to protonate the nucleus forming the molecular ion. In the second step, low-energy collision-induced dissociation tandem mass spectrometric (CID-MS/MS) was used to point the fragment, which shows the loss of dimethylamine. The data of the experiment did in fact show the observed mass of all 23 pregnane-type steroidal alkaloids, and when compared to known values, the percent errors were single digits. Using CID-MS/MS was extremely helpful into generating data that are useful for proposing a fragmentation pathway for the functional groups (see figure 11). [21]
Taken from [21]
Similar to every analytical technique in the world today, no single technique is able to provide answers about the characterization of a compound. ESI-MS has some advantages and some disadvantage. Electrospray ionization is a soft ionization technique which does not use a lot of energy, and as a result the sample being analyzes does not undergo through many fragmentations.[4] This is an advantage as more scientists are looking for instruments that will give them some conclusive information about the molecular weight of the large molecule. This leads to the second advantage is that a molecular ion is visible and it is so because ESI-MS is highly sensitive with good specificity.[5]Even though ESI-MS gives information about proteins such as molecular ions, there are cases where getting the molecular ion of some long proteins is not so easy. There have been reported cases where the molecular ion of some proteins were not visible, and that lowers the use of ESI-MS. Another disadvantage of ESI-MS is on the detection of peaks corresponding to dimeric species; conclusive evidence cannot be made by ESI-MS to show if dimeric species are present or not in a sample. [22] Also, in some sample, the peaks were not strong signals and that draws scientists a little back, as they are not sure about the nature of the peak. So, after stating the advantages and disadvantage of ESI-MS, it can be said that the technique is very useful in the characterization of compounds. To enhance this technique many scientists are combining this technique to other ones for better results.
In conclusion, electrospray ionization is the most commonly used analytical technique used by chemists and biochemist. In this paper, many examples of the use of ESI-MS in different fields have been reported to introduce the many different ways that this technique has been adopted. It is believed by many scientists that ESI-MS is still maturing, which leads us into hoping that someday, in the near future, ESI-MS can helps us identify and characterize with higher precision different types of compounds.[22] Electrospray ionization mass spectroscopy, similar to other analytical technique has both advantages and disadvantages to it, but looking at the information is has helped us gain so far, I strongly believe that its advantages overcome disadvantages. We are able to see its contributing when it is combined with other technique such as quadrupole time-of-flight, HPLC, and MS/MS. [6]
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